The most widely used sperm sorting methods generally rely on the detection of quantifiable differences in the DNA content of X-chromosome bearing sperm and Y-chromosome bearing sperm. Various modifications to flow cytometers for this purpose have been described in U.S. Pat. Nos. 5,135,759, 6,263,745, 7,371,517 and 7,758,811, each of which are incorporated herein by reference. In many species, this difference in DNA content can be small. In bovine, for example, Holstein bulls have about a 3.8% difference in DNA content, while Jersey bulls have about a 4.1% difference. The inexact nature of stoichiometric DNA staining makes these small differences difficult to ascertain and requires exposing sperm to damaging conditions over periods of time.
While Hoechst 33342 can be used in non-toxic concentrations, sperm must be incubated at elevated temperatures and elevated pHs for sufficient Hoechst 33342 penetration with sufficient uniformity for analysis or sorting. Each of elevating sperm temperature and changing the sperm pH may contribute to sperm damage. Additionally, the pressure and sheering forces applied to sperm cells within a flow cytometer may further compromise sperm membranes. These factors may accelerate the deterioration of sperm cell membranes further reducing the already limited shelf life of viable sperm.
Accordingly, previous sperm sorting efforts focused on utilizing smaller insemination samples and producing the greatest amount of sorted sperm in the shortest amount of time. U.S. Pat. No. 6,149,867, incorporated herein by reference, describes methods and devices geared towards helping sperm better survive flow cytometric sorting in combination with reduced dosage inseminates. Subsequent advances in flow sorting focused on improvements in detection or throughput. However, as speeds and throughputs increased, larger quantities of sperm, including viable sperm of the desired sex, are discarded with waste. Additional tradeoffs between purity and recovery also exist. For example, where the desirable purity is greater than 95%, fewer sperm can be determined with the requisite confidence level as compared to 70% 80% or 90% purities, meaning fewer sperm are sorted at increasingly high purities and that more viable sperm cells are disposed with the waste stream.
Additional losses in efficiency exist with respect to discarding viable sperm cells due to the occurrence of coincident events. A coincident event occurs when two or more sperm cells are too close together to be separated. In either event, all of the sperm cells may be discarded with waste, whereas some or all of those discarded cells may have been desirable to collect.
Previously, recovery problems were often overlooked, or moot, in view of raw flow sorting throughput. Bovine sperm, for example, is relatively easy to collect and process and high purities may be desirable in both the beef and dairy industries, even at the expense of discarding as much as about 90% of the sperm. However, this high throughput methodology is not acceptable for sperm in limited supply. For example, a specific animal could possess exceptionally desirable genetic qualities, but may produce poor sperm samples for sorting. A species could be rare, endangered, or difficult to collect, limiting the amount of sperm available for sorting. A previously collected sample may be preserved, but the animal or species may no longer be available for subsequent collections. Regardless of the circumstances, the wasteful sperm sorting process is undesirable for sperm in limited supply or sperm with high value. A need, therefore, exists for a method of sorting viable sperm with an improved efficiency in recovering sperm cells.